Analysis of Copy Number Variations in Patients with Autism Using Cytogenetic and MLPA Techniques: Report of 16p13.1p13.3 and 10q26.3 Duplications.

Autism is a common neuropsychiatric disorder affecting 1 in 68 children. Copy number variations (CNVs) are known to be major contributors of autism spectrum disorder (ASD). There are different whole genome or targeted techniques to identify CNVs in the patients including karyotyping, multiplex ligation-dependent probe amplification (MLPA) and array CGH. In this study, we used karyotyping and MLPA to detect CNVs in 50 Iranian patients with autism. GTG banding and 4 different MLPA kits (2 subtelomeric and 2 autism kits) were utilized. To elevate our detection rate, we selected the sporadic patients who had additional clinical features including intellectual disability, seizure, attention deficit hyperactivity disorder, and abnormal head circumference. Two out of 50 patients (4%) showed microscopic chromosome abnormalities and 5 out of 50 (10%) demonstrated copy number gains or losses using MLPA kits. Including one overlapping result between karyotype and MLPA techniques, our overall detection rate was 6 out of 50 (12%). Three out of 6 CNVs were de novo and three others were paternally inherited. Two of CNVs detected by karyotyping and MLPA tests were 16p13.1q13.3 and 10q26.3 duplications, respectively. For these two CNVs genotype and phenotype of the patients were compared with other studies. Although the pathogenicity of cytogenetic results was certain, most of MLPA results needed to be better refined using other more accurate techniques such as array CGH. Our findings suggest that it might be possible to obtain some useful information using MLPA technique but it cannot be used as a single diagnostic tool for the autism.

novo and three others were paternally inherited. Two of CNVs detected by karyotyping and MLPA tests were 16p13.1q13.3 and 10q26.3 duplications, respectively. For these two CNVs genotype and phenotype of the patients were compared with other studies. Although the pathogenicity of cytogenetic results was certain, most of MLPA results needed to be better refined using other more accurate techniques such as array CGH. Our findings suggest that it might be possible to obtain some useful information using MLPA technique but it cannot be used as a single diagnostic tool for the autism. Copy number variants are known to be major contributors with the overall rate of 10-15% in children with autism from which 3-7% are cytogenetically detectable chromosome abnormalities (5)(6)(7)(8) and the remaining could be identified using molecular cytogenetic techniques (9,10).
This rate increases when autism co-occurs with other clinical features, suggesting a syndromic forms of autism (11)(12)(13)(14)(15). Furthermore, de novo CNVs appear to be a more common risk factor in sporadic cases of ASD (12,16).
Almost every chromosome has been demonstrated to be involved in imbalances contributing to autism (8,17). Although most variants are very rare and might only be reported in a single case, several chromosomal abnormalities have been recurrently reported (5 (23)(24)(25).
The most commonly used methods to detect subtelomeric and interstitial chromosomal rearrangements include GTG banding, fluorescence in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), and microarray-based comparative genomic hybridization (array-CGH) (18). Out of these, cytogenetic is a cost-effective test that can detect all balanced and unbalanced rearrangements but with a size of at least 3-5 Mb.
MLPA is a targeted but rapid and cost-effective method to screen subtelomeric abnormalities and recurrent copy number changes involved in autism (26).
In the present study, our purpose was to evaluate the diagnostic efficiency of cytogenetic N and MLPA techniques in patients with autism. With regard to the finding that the highest occurrence of CNVs is demonstrated in singleton families (12,16) and when ASD is associated with additional clinical features, we used these parameters in the present study to elevate the detection rate. Fifty sporadic patients in whom clinical diagnosis was confirmed for amtism were selected for the present study. All and also in the SHANK2, an autism candidate gene.

Subjects
Clinical evaluation was carried out for the patients with ASD diagnosis. Fifty patients who met the DSM5 criteria diagnosed by pediatric neurologists specializing in autism were selected.
All patients were sporadic with no family history of ASD. The selected patients had ID with one or more additional clinical features.

GTG banding
High resolution GTG banding technique was carried out using standard protocols.

MLPA
The following 4 kits were used: SALSA

Results
Two out of 50 patients demonstrated chromosome abnormalities (4%) including one duplication on chromosome 16p and one deletion on chromosome 15q. Furthermore, all patients were investigated using 4 MLPA kits (Table 1). In one patient, different results were obtained using the subtelomeric kits (P036 and P070) that was a consequence of different genomic location of probes in two probemixes. As a result, copy number changes were demonstrated in 4 (8%) and 3(6%) patients using P036 and P070 kits, respectively. Utilizing the P343 kit, two (4%) CNVs were observed in the patients. However, none of the patients showed SHANK2 deletions with the P396 kit. Since some of the results were overlapping (Table 2), the total detection rate utilizing cytogenetic and MLPA analysis was 12% and MLPA showed a detection rate of 10%. Out of 6 abnormalities observed in the patients, 3 were de novo and 3 were paternally inherited. Additional clinical features presented in autism patients with copy number changes are shown in Table 3.    Both patients 5 (p35) and 6 (p36) whose clinical manifestations are shown in Table 3